Finding Lead Compounds for Dengue Antivirals from a Collection of Old Drugs through In Silico Target Prediction and Subsequent In Vitro Validation

Abstract

Dengue virus (DENV) infection is one of the most widely spread flavivirus infections. Despite the fatality it could cause, no antiviral treatment is currently available to treat the disease. Hence, this study aimed to repurpose old drugs as novel DENV NS3 inhibitors. Ligand-based (L-B) and proteochemometric (PCM) prediction models were built using 62,354 bioactivity data to screen for potential NS3 inhibitors. Selected drugs were then subjected to the foci forming unit reduction assay (FFURA) and protease inhibition assay. Finally, molecular docking was performed to validate these results. The in silico studies revealed that both models performed well in the internal and external validations. However, the L-B model showed better accuracy in the external validation in terms of its sensitivity (0.671). In the in vitro validation, all drugs (zileuton, trimethadione, and linalool) were able to moderately inhibit the viral activities at the highest concentration tested. Zileuton showed comparable results with linalool when tested at 2 mM against the DENV NS3 protease, with a reduction of protease activity at 17.89 and 18.42%, respectively. Two new compounds were also proposed through the combination of the selected drugs, which are ziltri (zilueton + trimethadione) and zilool (zileuton + linalool). The molecular docking study confirms the in vitro observations where all drugs and proposed compounds were able to achieve binding affinity ≥ -4.1 kcal/mol, with ziltri showing the highest affinity at -7.7 kcal/mol, surpassing the control, panduratin A. The occupation of both S1 and S2 subpockets of NS2B-NS3 may be essential and a reason for the lower binding energy shown by the proposed compounds compared to the screened drugs. Based on the results, this study provided five potential new lead compounds (ziltri, zilool, zileuton, linalool, and trimethadione) for DENV that could be modified further.

Figure 1

Applicability domain (AD) using similarity search and leverage. Based on the score, both algorithms show that the model reliably predicts the test set activity. Analyses were conducted using the Knime Analytics Platform.

Figure 2

Vability of Vero cells after treatments with different drugs: (A) zileuton, (B) trimethadione, (C) linalool, and (D) positive control—ribavirin. The viability of cells was analyzed at 72 h using the MTT assay. No significant cell morphology changes were detected at the highest concentration tested. The CC₅₀ values are expressed as percentages of treated vs. untreated cells. Each value is the means ± SD of three experiments, each run in triplicate.

Figure 3

Inhibitory potential of drugs on infected cells with the dengue virus: (A) zileuton, (B) trimethadione, (C) linalool, and (D) positive control—ribavirin. The drug was diluted to various concentrations (as indicated above) with overlay media and added to the cell culture. The viral inhibition was analyzed at 72 h. The inhibitory response of each drug is expressed as the mean of absorbance ± SD of three experiments, each run in triplicate. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4

NS2B-NS3 protease inhibition with the treatment of selected drugs: (A) zileuton, (B) trimethadione, (C) linalool, (D) positive control, aprotinin. and (E) positive control—ribavirin. The statistical analysis was conducted between particular drugs against the untreated cells (NC) using one-way ANOVA with Tukey correction. Each value is the means ± SD of two experiments, each run in triplicate. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Newly developed compounds based on the in vitro results. (A) Ziltri (zileuton + trimethadione) and (B) zilool (zileuton + linalool).

Figure 6

Molecular surface representation of the NS2B-NS3 protease with selected drugs. Selected drugs from two prediction models (zileuton, trimethadione, linalool), newly developed compounds (ziltri and zilool) and positive control—panduratin A, were docked at the active site of the protease.

Figure 7

Interactions of the selected drugs and substances against the dengue NS2B-NS3 protease both in 3D and 2D visualizations. The complex of the NS2B-NS3 protease with zileuton (A, B), trimethadione (C, D), linalool (E, F), ziltri (G, H), zilool (I, J), and panduratin A (K, L).

Figure 8

Workflow of the study. The study begins with building a prediction model that could screen marketed drugs for activity against the NS3 dengue protein. Here, ligand-based (LB) and proteochemometric (PCM) models were developed. Next, the in vitro validations were conducted to analyze the antiviral properties of the drugs. Here, the cytotoxicity assay, FFURA, and protease inhibition assay were conducted. The final stage of the evaluation was done to determine factors that possibly contribute to the interaction observed in vitro using molecular docking. Here, molecular docking was done on the selected drugs against the NS3 protease. The binding energy and the interactions that developed between the ligand–protein complexes were analyzed.